Fig 3.

SFRP1 downregulation alleviates etoposide-induced senescence. (A) shRNA knockdown of SFRP1. IMR-90 cells were infected with lentiviruses expressing shRNA against luciferase (shLuc; control) or shRNAs against SFRP1 (shSFRP1-1 and shSFRP1-3) and were selected with 2 μg/ml puromycin. Five days after infection, conditioned medium was collected and was analyzed for SFRP1 and SPARC levels by immunoblotting. (B and C) SFRP1 knockdown attenuates etoposide-induced senescence. The effect of SFRP1 knockdown on etoposide-induced senescence of IMR-90 cells was analyzed 6 days after etoposide treatment. (B) SA-β-Gal staining. (C) Senescence-associated heterochromatic foci (SAHF). A minimum of 100 cells were counted. *, P < 0.05 compared to shLuc plus 20 μM etoposide. (D) SFRP1 antibody inhibits etoposide-induced senescence. IMR-90 cells were treated for 48 h with etoposide. Beginning 24 h after treatment, the cells were incubated with either 1 μg/ml of control rabbit IgG, anti-SFRP1 antibody (rabbit polyclonal H-90; Santa Cruz Biotechnology), or anti-SFRP1 antibody (Ab) plus immunogen (block [B]). Cells were assayed for SA-β-Gal staining 5 days after etoposide treatment. *, P < 0.05 compared to etoposide plus the IgG control or to etoposide plus anti-SFRP1 and block. (E) SFRP1 antibody does not affect Ras-induced senescence. IMR-90 cells were infected with c-H-RasV12-expressing lentivirus (empty vector lentivirus as a control). Beginning 4 days after infection, the cells were incubated with either 1 μg/ml of control rabbit IgG or anti-SFRP1 antibody. Eight days after infection, the cells were stained for SA-β-Gal. (F) SFRP1 knockdown abolishes oxidative stress-induced senescence. IMR-90 cells were treated with 500 μM H₂O₂ for 2 h. The following day, the cells were infected with lentiviruses expressing shRNA against luciferase or SFRP1. On day 6 after H₂O₂ treatment, the cells were analyzed for SA-β-Gal. *, P < 0.05.